Many of physiologically-active substances have so-called chirality. Examples of such physiologically-active substances are a medical substance, a poisonous substance, and a functional component in a living organism. It is widely known that physiological activities of such substances strongly depend on chirality, that is, high-order structures (conformations, configurations, and the like) of the substances. Thus, it is important in a study of physiologically-active substances to understand chirality of the substances. In this regard, chirality and high-order structures of biomolecules including protein, nucleic acid, and the like, in particular, have been spotlighted in recent years.
These biomolecules are thought to dynamically change their structures in living organisms. For example, an HIV is known to cause AIDS infection by protease that sandwiches and cuts protein of a host. The protease is thought to have a significant change in its conformation when cutting protein. Moreover, a significant change in a high-order structure of prion protein is believed to cause Bovine Spongiform Encephalopathy (BSE).
As is obvious from the above description, analysis of high-order structures of biomolecules such as protein and the like is effective in treatment and diagnosis of diseases. For this reason, techniques for analyzing high-order structures of biomolecules have been developed. Typically known techniques for such analysis are circular dichroism (CD) spectrum analysis, fluorescence detected circular dichroism (FDCD) spectrum analysis, circularly polarized luminescence dichroism (CPL) spectrum analysis, and the like.
For example, Patent Document 1 discloses a technique that relates to the FDCD. Specifically, the Patent Document 1 discloses a method of analyzing chirality which method includes the steps of: introducing a fluorescent functional group to a substance having chirality; exciting the fluorescent functional group by irradiating the group with a right circularly polarized light and a left circularly polarized light and measuring fluorescence intensities that are obtained by excitation with the use of the left and right circularly polarized lights, respectively; and analyzing the chirality of the substance based on difference information between the fluorescence intensities that are obtained by excitation with the use of the right circularly polarized light and excitation with the use of the left circularly polarized light, respectively.
Moreover, Patent Document 2 discloses an apparatus for measuring a circular dichroism fluorescence excitation spectrum which apparatus (i) measures fluorescence intensities obtained by irradiating a sample alternately with the use of a right circularly polarized light and a left circularly polarized light at a predetermined modulation frequency, which circularly polarized lights are for wavelength scanning and homochromatic, and, then, (ii) converts the fluorescence intensities into electrical signals. Patent Document 2 discloses that, in the apparatus, detection sensitivity is improved by independently using, out of the electrical signals, only an alternating-current signal component in synchronization with a frequency for switching between the right and left circularly polarized lights, so as to obtain a circular dichroism fluorescence excitation spectrum.
Furthermore, Patent Document 3 discloses a method and an apparatus for measuring a CPL or an FDCD of a sample by using a laser scanning microscope. For example, FIG. 4 of Patent Document 3 is a diagram schematically illustrating an apparatus for measuring a CPL, while FIG. 9 is a diagram schematically illustrating an apparatus for measuring an FDCD.
(Patent Document 1)
Japanese Unexamined Patent Publication No. 2606/1999 (Tokukai-hei 11-2606) (published on Jan. 6, 1999)
(Patent Document 2)
Japanese Unexamined Patent Publication No. 23466/1999 (Tokukai-hei 11-23466) (published on Jan. 29, 1999)
(Patent Document 3)
USP Application Publication No. 2003/0058442 A1 (published on May 27, 2003)
Chirality of a biomolecule such as protein or the like is believed to depend, to a large extent, on an environment in which the biomolecule exists. For example, it is predicted that protein, nucleic acid, or the like change a conformation thereof, interacting with other protein, a cell membrane, or the like.
However, it is impossible to analyze such a phenomenon in a uniform experimental system in a test tube. Thus, the inventors of the present invention judged that, in analysis of a high-order structure of a biomolecule, it was very important to analyze the structure directly in a living organism (e.g., in a cell or the like). However, all of the aforesaid techniques such as the CD, the FDCD, and the CPL are designed to analyze chirality of substances that exist in uniform solutions. Therefore, these techniques are not suitable for analyzing chirality of substances that exist in non-uniform environments, such as in living organisms, where various substances exist.
Moreover, the technique disclosed in each of the Patent Documents 1 and 2 is not directed to a microscope analysis, and requires a large amount of a sample for analysis. However, generally, only a very small amount of a biomolecule can be prepared. Accordingly, it takes much time and cost to obtain such a large amount of the sample.
Further, according to the technique disclosed in the Patent Document 3, it is highly likely that measurement is not possible at a practical level. Particularly, according to the technique disclosed in the Patent Document 3, it is impossible to analyze a sample that emits light whose circularly polarized light component is small.
In addition, in the technique disclosed in the Patent Document 3, a circularly polarized luminescence dichroism (CPL) spectrum analysis is carried out with the use of a semi-transmissive mirror that serves as means for transmitting an excitation light and reflecting fluorescence light. However, the semi-transmissive mirror (i) lacks wavelength selectivity and (ii) is semi-transmissive. Thus, a loss of light intensity of the incident excitation light occurs. Further, a loss of light intensity of the reflected fluorescence light also occurs. Such losses cause a problem of significant deterioration in measurement accuracy. Moreover, unless the semi-transmissive mirror is arranged exactly at 45 degrees with respect to a light path, distortion occurs. This also results in a problem of significant deterioration in measurement accuracy and stability.
Furthermore, in the technique disclosed in the Patent Document 3, as illustrated in FIG. 4, an iris is disposed near a detecting section. This arrangement causes a problem such that an error occurs in detection of a polarized light component of fluorescence light because the fluorescence light that is emitted from the sample is multi-reflected within a polarization modulating section.
Therefore, it has been strongly desired to develop a technique in which (i) a large amount of a sample is not required for analysis and (ii) a high-order structure of a biomolecule such as protein or the like can be analyzed with high accuracy directly in a living organism. However, research and development of such a technique has not been carried out conventionally. Accordingly, development of the aforesaid technique that contributes to exploitation of novel fields is strongly desired.
The present invention is attained in view of the problem. An object of the present invention is to provide a circular dichroism fluorescent microscope that does not require a large amount of a sample for analysis and that, for example, can analyze, with high accuracy, a high-order structure of a sample including a biomolecule or the like such as protein, directly in a living organism.